Substances existing in living organisms play various roles, and thus biological reactions also have a wide variety of patterns. The term “biological reactions” used herein means enzymatic reactions for performing degradation, transition, addition or synthesis on a protein, nucleic acid, lipid, carbohydrate or the like, and also changes in the concentrations of substances in vivo, changes in intracellular localizations, etc. Changes occurring in living organisms/cells are caused by a series of temporal or spatial chain reactions promoted by multicomponent constructs in many cases. Accordingly, by simultaneous monitoring such reactions promoted by various living organism constituents, it becomes possible to grasp the conditions of living organisms/cells more strictly. It is anticipated that information obtained as a result of such monitoring will be useful indicators in drug discovery. However, currently, neither a method of monitoring reactions of different patterns promoted by different components, nor even a method of monitoring multiple enzymatic reactions, has been developed.
The term “strictly” is used herein to mean not only a detailed analysis of changes in the conditions of organisms or cells, but also a case where internal standard reactions established must be simultaneously measured to conduct quantitative comparative discussion in diagnosis or the like, beyond differences among sample preparations, measurement apparatuses, and facilities, and where other types of reactions must be simultaneously measured as internal standard reactions even in the case of using a certain enzyme activity for the diagnosis of pathologic conditions.
A majority of products, called protein chip, are able to detect the amount (expression level) of a target protein using an antibody, just same as the ELISA method. However, a protein chip like that cannot reveal a reaction mechanism such as a detailed interaction mechanism, with which the protein is associated in a living body. In addition, even in identification of a phosphorylated protein using mass spectrometry (MS), protein chip like that is used only for confirmation of the amount of such a modified protein. That is to say, the activity of a modifying enzyme cannot be grasped using protein chip like that.
Moreover, many artificial substrates have been developed for the measurement of enzyme activity. However, it is difficult to align such artificial substrates in parallel and to use them in a high throughput manner. Since in prior art techniques, the used reagents are similar even in a case where targets to be measured are completely different, different types of activities cannot be simultaneously measured in most cases.
Hence, the present inventors have performed a protein engineering modification on GFP. Thereafter, the inventors have bound a GFP to a fluorescent dye or to a biomolecule labeled with such a fluorescent dye through introduced new amino acid sequence or the like, so as to prepare a complex molecule (bioprobe). Thus, the inventors have developed a method of monitoring various reactions promoted by living organism constituents in a parallel manner. This is a method, which comprises converting Fluorescence Resonance Energy Transfer (FRET) observed between GFP (or other types of fluorescent proteins) and a fluorescent dye (or a biomolecule labeled with such a fluorescent dye) that forms a complex with fluorescent protein in a single fusion molecule or in two closely existing molecules, into changes in states of biological reactions, and then monitoring FRET regardless within or without cells (e.g. Non-Patent Document 1, Non-Patent Document 2, and Patent Document 1). The existing protease analysis method is almost specialized in the analysis of amount. In contrast, this methodology is considered excellent one that is able to monitor reactions themselves or changes in states. Moreover, it has been revealed that such reactions or changes in states could be monitored not only by FRET but also by Fluorescence Cross-Correlation Spectroscopy (FCCS) observed between two fluorescent molecules.
Furthermore, an analysis method using FRET on a bioprobe constituted with a quantum dot and a biomolecule labeled with a fluorescent molecule has also been reported to date. However, for such a reason as difficulty in the measurement of substantial FRET and FRET dependent on a measurement target, precise measurement has not yet been achieved in the method (Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5).
[Patent Document 1] Japanese Patent Application Laid-Open (Kokai) No. 2002-153279
[Patent Document 2] WO2004/042404
[Non-Patent Document 1] Suzuki et al., Biochim. Biophys. Acta, 1679: 222-229, 2004
[Non-Patent Document 2] Suzuki et al., Biochim. Biophys. Res. Comm., 330: 454-460, 2005
[Non-Patent Document 3] Chang et al., Biochem. Biophys. Res. Comm., 334: 1317-1321, 2005
[Non-Patent Document 4] Lin et al., Anal. Biochem., 319: 239-243, 2003
[Non-Patent Document 5] Medintz et al., Proc. Nat. Acad. Sci. USA, 101: 9612-9617, 2004